Abstract Human T-cell leukemia virus (HTLV-1) infects about 15-20 million individuals worldwide and is the etiological agentofanadultT-cellleukemia/lymphoma(ATLL),andcanalsoresultinaninflammatorydiseasesyndrome calledHTLV-1-associatedmyelopathy(HAM)/tropicalspasticparaparesis(TSP).HTLV-1antibodyprevalence rates vary among geographical areas, ranging from 0.2 to 10% among adults. This antibody prevalence increaseswithage,andcanaffectasmuchas20to50%ofthefemalepopulationaged60andabove.HTLV-1 is notorious for being difficult to study in cell culture, which has prohibited a rigorous analysis of how these viruses replicate in cells, including the steps involved in retrovirus assembly. The details for how retrovirus particle assembly occurs are poorly understood even for other more tractable retroviral systems like that of human immunodeficiency virus type 1 (HIV-1). For instance, recent evidence indicates that Gag-Gag interactionsdifferamongretroviruses,whichhelpsexplainmorphologicaldifferencesthatwehavedocumented amongimmatureretrovirusparticles.Furthermore,theroleformembrane-bound,non-punctate(np)Gaginthe biogenesisofGagpuncta,aswellasthenatureofGagpunctabiogenesisinthecontextofcell-to-cellcontacts alsoremainpoorlyunderstoodaspectsoftheretrovirusassemblypathway.ThisisparticularlyforHTLV-1,for which we have found to have fundamentally distinct differences to that of HIV-1 regarding the role of membrane-bound np Gag in Gag punta biogenesis. In this application, we propose to continue our investigations on HTLV-1 immature and mature particle structure and particle biogenesis through innovative state-of-the-artexperimentalapproacheswithappropriatecomparativeanalyseswithHIV-1.Inparticular,we willapplycryo-electronmicroscopy/tomography(cryo-EM/ET),photoactivatedlocalizationmicroscopy(PALM), total internal reflection fluorescence (TIRF) microscopy and the novel technology of z-scan fluorescence fluctuation spectroscopy (FFS) in living cells to investigate 1) analysis of immature HTLV-1 Gag lattice structure, 2) the importance of membrane-bound, np Gag in HTLV-1 particle biogenesis, and 3) HTLV-1 particle biogenesis in the context of cell-cell contacts. Careful comparisons will be done with HIV-1. These novel studies harness innovative technologies in order to provide new insights into a highly significant and poorlyunderstoodaspectoftheHTLV-1particleassemblyprocess,whichisfundamentallydistinctfromthatof HIV-1andotherretroviruses.Furthermore,ourstudiesrepresentsomeofthemostdetailedstudiesconducted ontheassemblyofvirusstructuralproteins,whichprovidesfundamentallyimportantinformationinvirologyon themolecularnatureofvirusparticleassemblyattheplasmamembraneforenvelopedviruses.